Despite tremendous strides in transplantation over the past 60 years, with great advances in short-term graft survival, long-term survival is comparatively poor. This is likely due to the fact that immunosuppressive strategies, though clearly improved over the years, are not tolerogenic. True transplantation tolerance is likely to arise not from improved immunosuppression, but from developing novel strategies based on improved understanding of the physiologic mechanisms which maintain self-tolerance, and the barriers that mediate resistance to tolerance. Moreover, approaches for transplantation tolerance are likely to be applicable for the treatment of many autoimmune disorders as well. Our approach is based on the premise that the mechanisms which mediate peripheral self-tolerance may prove most easily exploited to generate tolerance alloantigens in adults. The overall theme is that the availability of novel t cell activation/differentiation signals, the cytokine milieu, and the presence or absence of inflammation collectively determine whether T cells will be directed towards regulation or effector/memory functions, and thus these parameters determine the ultimate outcome of the alloimmune response; rejection versus tolerance. The aims of project #1 are designed to test the central hypothesis that the TIM-1 :TIM-4 pathway, by affecting Th1/Th2 cell differentiation, generation of effector/memory T cells, and possibly regulatory T cell generation and function, plays an important role in alloimmune and autoimmune responses and tolerance in the setting of islet transplantation. The aims of project #2 are focused on the concept that two events occurring very early during the process of engraftment, inflammation and antigen encounter, determine graft outcome and tolerance susceptibility by modulating the balance between regulatory cells and effector/memory cells. Lastly, project #3 will study how the cytokine milieu determines the balance between the development of pathogenic T cells and tolerogenic regulatory cells. Together, these investigations should provide new insights into how alloimmune T cell responses are initiated and controlled. The results should yield useful new information that can be harnessed to develop novel tolerogenic strategies to test in primates and ultimately humans. PROJECT 1: The Role of Novel T Cell Costimulatory Pathways in Allograft Rejection and Tolerance (Sayegh, M.) PROJECT 1 DESCRIPTION (provided by applicant): The T cell immunoglobulin mucin (TIM) family of novel receptor-ligand pairs plays important roles in T cell activation, differentiation and effector/memory function, and in regulation of immune responses in autoimmunity and allergy/asthma. TIM-1 is expressed by activated Th1 and Th2 cells and its expression is sustained preferentially in terminally differentiated Th2 cells. The ligand for TIM-1 is TIM-4, which is predominantly expressed on APCs. Recent studies indicate that TIM-1 may differentially regulate T helper cell (Th1/Th2) differentiation in asthma/allergy, and autoimmune encephalomyelitis. At present, little is known about the role of the TIM-1 :TIM-4 pathway in alloimmune responses and autoimmune diabetes. Preliminary studies from our group indicate that the TIM-1 :TIM-4 pathway plays an important role in alloimmunity, particularly alloreactive T helper cell differentiation and possibly regulatory T cell generation/function. Furthermore, it is well established that the balance of autoreactive Th1 cells on one hand and regulatory T cells and Th2 cells on the other is critical in determining the outcome of autoimmune diabetes in NOD mice. Our central hypothesis is that the TIM-1 :TIM-4 pathway, by modulating Th1/Th2 cell differentiation and possibly regulatory T cell generation and function, plays an important role in alloimmune and autoimmune responses, and tolerance. The main goal of this proposal is to define the functions and mechanisms of the TIM-1 :TIM-4 pathway in regulating immune responses in vivo as a means of developing novel strategies to achieve durable and reproducible tolerance, and preventing the development of recurrent autoimmunity to islet allografts. In that regard, our approach is to test and explore the mechanisms of novel rational combination strategies that target multiple pathways resulting in silencing of alloreactive and autoreactive T cells, and tipping the balance towards regulation by cells and/or cytokines in NOD recipients of islet allografts. In Specific Aim 1 we will investigate the effects of targeting the TIM-1 :TIM-4 pathway on alloimmune and autoimmune responses in vivo in models of islet allograft rejection. In Specific Aim 2 we will dissect the mechanisms of action of TIM-1 :TIM-4 pathway in alloimmunity, autoimmunity and tolerance, focusing on T cell expansion, differentiation, and apoptosis. These studies will utilize CD4+ and CD8+ TCR transgenic animals with defined allo- (B6 background) and auto- (NOD background) specificities. MHC tetramers will also be used to study the mechanisms of targeting TIM-1 on autoreactive CD4+ and CD8+ T cells in NOD mice. Finally, in Specific Aim 3 we will focus specifically on the role of TIM-1 :TIM-4 pathway in the generation and/or function of regulatory T cells in vivo. Using foxp3-GFP knock-in reporter mice on B6 and NOD backgrounds, we will test the hypothesis, based on initial preliminary data, that the TIM-1 :TIM-4 pathway may have an important role in the generation and/or function of CD4+CD25+ regulatory T cells in vivo. Overall, our studies should yield useful new data that may lead to development of novel strategies to induce tolerance to islet allografts to translate to primates and humans.